Melanoma is a devastating form of skin cancer with limited therapeutic options. This project focuses on the signaling pathways in NRAS mutant melanomas, which accounts for approximately 20% of melanomas. Therapies that target RAS and MEK, a RAS effector, have not been clinical effective in mutant NRAS melanoma patients. In cell-based studies, we show that the response of NRAS mutant melanomas to the MEK inhibitor AZD6244 is heterogeneous, with a sensitive and an insensitive group. For the AZD6244 insensitive cells, there is a need to identify specific effectors that mediate mutant NRAS actions and additional pathways that are synergistic for NRAS signaling to form the basis of new therapeutic strategies. One protein that may prove to be important in this group is TANK-binding kinase 1 (TBK1), an atypical IkB kinase family member. In KRAS transformed cancer cells, TBK1 mediates oncogenesis through its phosphorylation and activation of the pro-survival kinase AKT. We show that NRAS expression increases TBK1 activation in melanoma cells. In mutant NRAS melanoma cells, we will determine if TBK1 regulates AKT activation, as measured by Western blot and kinase assays, through TBK1 depletion. To assess its potential as a therapeutic option, we targeted TBK1, via RNA interference or the pharmacological inhibitor BX795, in conjunction with MEK. We found that the combination leads to increased cell death in AZD6244 insensitive NRAS mutant melanoma cells. We have obtained a more specific inhibitor of TBK1, Compound II, and we will use it alone and in combination with AZD6244 in in vitro 3D collagen survival assays (which mimic the dermal environment) as well as in in vivo intradermal xenografts. In these experiments, we will be utilizing melanoma cell lines as well as cells dissociated from surgically resected NRAS mutant tumors. Additionally, we will analyze expression and activation of TBK1 in tumor microarrays of metastatic melanomas, associating our findings with NRAS genotype. In addition to TBK1, we predict that NRAS mutant melanoma cells also rely on genes that are not directly downstream of NRAS for survival. To identify signaling alterations in AZD6244 insensitive cell, we will use a short hairpin RNA (shRNA) library in a negative selection screen to find shRNA that are selectively toxic to AZD6244-treated AZD6244 insensitive cells. To validate the targets, we will knockdown the target using RNA interference to see if the lethal phenotype can be recapitulated. The validated targets will be examined in AZD6244 sensitive and insensitive cells and overexpressed in AZD6244 sensitive cells to see if they confer resistance to MEK inhibition. If targets or their involved pathways have inhibitors, then we will examine the effects, either alone or in combination with AZD6244, on cell death in 3D collagen survival assays. The data generated from this proposal may lead to the identification of TBK1, along with other proteins, as novel targets for NRAS mutant melanoma to be used in the clinical setting as part of a combination therapy regimen. Our long-term objective is to improve the treatment options for NRAS mutant melanomas. PUBLIC HEALTH RELEVANCE: This project seeks to elucidate the molecular mechanisms underlying melanomas with a NRAS mutation. The results could lead to the identification of a novel therapy regimen for patients with NRAS mutant melanoma.